The present invention relates to genes isolated from stigma tissues. More specifically, the present invention relates to a gene and regulatory region isolated from corn silk.
The silk is part of the female reproductive organ in corn. It is a long hairy structure attached to the ovary. Pollen grains lodge upon the silk, they germinate and the pollen tubes grow down the hair into the silk to reach the ovules (Bonnett, O. T., 1948). The corn silk corresponds to the stigma in typical flowers (Heslop-Harrison et al, 1984). The stigma is responsible for capturing and selecting compatible pollen grains and for facilitating their germination.
The silk strands are mainly constituted of one external epidermal layer surrounding many cell layers of parenchyma tissues, and of vascular bundles located within the parenchyma tissue. The vascular bundle areas include pollen transmitting tissues, xylem elements, sieve tube elements and companion cells. The companion cells are morphologically and physiologically associated with the sieve tubes elements. Companion cells are key players in the phloem loading of assimilates and the synthesis of proteins targeted to enucleate sieve tubes and their functions require transport through plasmodesmata to sieve elements.
Genes and promoters that have been shown to function primarily in the stigma have been identified primarily in a few dicot species. They include proteinase inhibitors from Nicotiana alara (Atkinson et al, 1993), chitinases from Petunia hybrida (Leung, 1992) and genes involved in the sporophytic self-incompatibility system of Brassica (Dzelzkalns et al, 1993; Goring et al, 1993; Nasrallah and Nasrallah, 1993; Robert et al, 1994; Trick and Heizmann, 1992). A gene from soybean, ENOD40(2) has been found to function in root and stigma (Mirabella et al, 1999).
A number of genes have been shown to be preferentially expressed in the stigma of Brassica that correspond to genes associated with SI: SLG (S-locus glycoprotein), SRK (S receptor kinase; U.S. Pat. No. 5,484,905) or SLR (S-locus-related; WO94/25613) genes (for review: Nasrallah, J. B., Nasrallah, M. E. Plant Cell 5:1325-1335 (1993)). SRK6 is reported to be preferentially expressed in pistil and anther tissues (U.S. Pat. No. 5,484,905). U.S. Pat. No. 5,585,543 discloses the use of the S-gene for altering self incompatibility. The S3 promoter was found to confer expression within the pistil.
Another example of a gene expressed in the Brassica stigma is Pis 63 (U.S. Pat. No. 5,907,093; Robert, L. S. et al Plant Mol. Biol 26: 1217-1222 (1994)). The preparation of plants with female sterility based on a style-stigma specific xe2x80x9cSTMGxe2x80x9d gene and derived constructs using PSTMG promoter cassettes is disclosed in U.S. Pat. No. 5,633,441. These constructs include transcriptional fusions comprising barnase, papain or RNAse. Related U.S. Pat. No. 5,767,374 also discloses female sterile plants which are transformed with foreign DNA encoding a female sterility protein and a promoter which may be tissue specific, for example, a stigma-specific promoter. However, in both U.S. Pat. Nos. 5,633,441 and 5,767,374 no silk-specific promoters or their use, is disclosed. U.S. Pat. No. 5,723,763 discloses a method for identifying a tissue-specific promoter including stigma specific promoters.
An example of a promoter expressed in the companion cells of Arabidopsis thaliana leaves, stems and sepals is AtSUC2 (Truernit, E. and Sauer, N. Planta 196:564-570 (1995)). This promoter has low or undetectable activity in many other tissues of the transgenic plants, including petals, anthers and pistils.
The promoters identified above have been isolated from dicot plants. None of these promoters have been isolated from corn silk, nor is it known if these promoters may function in corn silk. U.S. Pat. Nos. 5,608,143 and 5,364,780 disclose nucleic acid promoter fragments from corn, however, no promoters are reported from silk. U.S. Pat. No. 5,589,610 discloses stamen specific promoters from corn. No stigma-specific promoters are disclosed.
The silk is also one of the two principal routes of invasion by the fungus Fusarium graminearum in corn. Fungal spores lodge upon the silk, germinate and the mycelia grow down the strands, either inside or outside, until they reach the ovules where the infection develops further. Expression of certain defence genes in silk may increase the resistance of corn to Fusarium species, however, no silk-specific promoters have been identified that could be used for this purpose. Furthermore, it is not known if stigma specific promoters isolated from dicot species such as Brassica napus would be functional in corn.
U.S. Pat. No. 5,859,351 discloses the Prf gene (from tomato) which encodes a protein that confers phytopathogenic resistance to transgenic plants against such pathogens as Pseudomonas and Xanthomonas. The use of silk- or monocot-specific promoters is not noted. U.S. Pat. No. 5,670,349 discloses a HMG2 promoter responsive to pathogen infection, pest infection and chemical induction that can be used to drive expression of disease and pest resistance genes of interest. The promoter is active in pollen and mature fruit tissues, however, there is no disclosure of whether it is active in corn silk.
WO 98/18939 discloses a nucleotide sequence encoding a salicylic acid-induced protein (SIP) kinase which may activate plant defences against microbial pathogens, and may be activated by, for example, a microbial pathogen, or an elicitor such as salicylic acid. Transformation of plants with this kinase enhances disease resistance to certain pathogens by inducing certain defence responses in a plant. The use of CaMV 35S is discussed, but tissue specific silk, or pistil/stigma, promoters are not discussed.
WO 99/02655 discloses nucleic acids encoding protein kinase genes, that can be induced by pathogen invasion or elicitor treatment. Plants transformed with such nucleic acids have enhanced pathogen resistance. Inducible regulatory elements flanking the nucleic acid may also be included in the transformed plant, however, no mention is made of silk or stigma specific promoters.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
The present invention relates to genes isolated from stigma tissues. More specifically, the present invention relates to a gene and regulatory region isolated from corn silk.
According to the present invention there is provided a regulatory region comprising an isolated nucleic acid obtained from corn silk.
This invention is also directed to an isolated nucleic acid comprising a silk-specific regulatory region or a fragment thereof, or a nucleic acid that is substantially homologous with the silk-specific regulatory region or a fragment thereof, wherein the nucleic acid that is substantially homologous with the silk regulatory region or fragment thereof, exhibits silk activity.
The present invention also provides for an isolated nucleic acid comprising nucleotides 1 to about 1986 of SEQ ID NO:2 or a fragment thereof, or a nucleic acid that is substantially homologous with nucleotides 1 to about 1986 of SEQ ID NO:2 or a fragment thereof, wherein the nucleic acid that is substantially homologous with nucleotides 1 to about 1986 of SEQ ID NO:2 or a fragment thereof, exhibits silk activity.
The present invention also pertains to an isolated nucleic acid comprising nucleotides 1 to about 1959, from about 208 to about 1986, from about 888 to about 1986, from about 1134 to about 1986, from about 1446 to about 1986, from about 1668 to about 1986, or from about 1868 to about 1986 of SEQ ID NO:2.
Furthermore, the present invention is directed to an isolated nucleic acid comprising nucleotides from about 1668 to about 1868 of SEQ ID NO:2.
This invention includes a chimeric construct comprising the regulatory region comprising an isolated nucleic acid obtained from corn silk in operative association with a gene of interest. It also is directed to a vector comprising the chimeric construct, and a transgenic plant comprising the vector.
The present invention is directed to a chimeric construct comprising an isolated nucleic acid obtained from corn silk in operative association with a heterologous regulatory element, to a vector comprising this chimeric construct, to a transgenic plant cell comprising this vector, to transgenic seed comprising this vector, and to a transgenic plant comprising this vector.
The present invention provides for a method of producing a female sterile plant comprising:
i) transforming a plant for which female sterility is desired with a regulatory region comprising an isolated nucleic acid obtained from corn silk, inoperative association with a gene that encodes a protein involved in inhibiting pistil development, pollen stigma interactions, pollen tube growth or fertilization, or a combination thereof to produce a transformed plant; and
ii) growing said transformed plant.
Also included within this invention is a method of producing a plant with increased pathogen resistance comprising:
i) transforming a plant with a regulatory region comprising an isolated nucleic acid obtained from corn silk, inoperative association with a gene that encodes a protein involved in inducing pathogen resistance, to produce a transformed plant; and
ii) growing said transformed plant.
The present invention is also directed to a method of producing a plant expressing a gene of interest within the pistil comprising:
i) transforming a plant with a vector, the vector comprising a regulatory region obtained from corn silk in operative association with a gene of interest, to produce a transforrned plant; and
ii) growing said transformed plant.
The regulatory region obtained from corn silk may be used to direct the expression of a gene of interest in the stigma from a range of plants. Furthermore, the activity of the regulatory region of the present invention may be modified by the presence of other regulatory regions, for example, enhancers, core promoter elements and the like, in operative association with a regulatory region of the present invention.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.